Non-human transgenic mammals expressing an n-3 desaturase gene

ABSTRACT

The present invention features compositions (e.g, nucleic acids encoding fat-1, optionally and operably linked to a constitutively active or tissue-specific promoter or other regulatory sequence and pharmaceutically acceptable formulations including that nucleic acid or biologically active variants thereof) and methods that can be used to effectively modify the content of PUFAs in animal cells (i.e., cells other than those of  C. elegants , for example, mammalian cells such as myocytes, neurons (whether of the periferal or central nervous system), adipocytes, endothelial cells, and cancer cells). The modified cells, whether in vivo or ex vivo (e.g., in tissue culture), transgenic animals containing them, and food products obtained from those animals (e.g., meat or other edible parts of the animals (e.g., liver, kidney, or sweetbreads)) are also within the scope of the present invention.

This application claims priority from U.S. Ser. No. 60/275,222, filedMar. 12, 2001, the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This invention relates to compositions and methods for altering thecontent of polyunsaturated fatty acids in mammalian cells.

BACKGROUND

Some of the work presented herein was supported by a grant from theNational Institutes of Health (CA79553). The United States governmentmay, therefore, have certain rights in the invention.

Polyunsaturated fatty acids (PUFAs) are fatty acids having 18 or morecarbon atoms and two or more double bonds. They can be classified intotwo groups, n-6 or n-3, depending on the position (n) of the double bondnearest the methyl end of the fatty acid (Gill and Valivety, TrendsBiotechnol. 15:401–409, 1997; Broun et al., Annu. Rev. Nutr. 19:197–216,1999; Napier et al., Curr. Opin. Plant Biol. 2:123–127, 1999). The n-6and n-3 PUFAs are synthesized through an alternating series ofdesaturations and elongations beginning with either linoleic acid (LA,18:2n6) or α-linolenic acid (ALA, 18:3n3), respectively (Gill andValivety, supra; Broun et al., supra; Napier et al., supra). The majorend point of the n-6 pathway in mammals is arachidonic acid (AA, 20:4n6)and major end points of the n-3 pathway are eicosapentaenoic acid (EPA,20:5n3) and docosahexaenoic acid (DHA, 22:6n3).

An important class of enzymes involved in the synthesis of PUFAs is theclass of fatty acid desaturases. These enzymes introduce double bondsinto the hydrocarbon chain at positions determined by the enzyme'sspecificity. Although, in most cases, animals contain the enzymaticactivity to convert LA (18:2n6) and ALA (18:3n3) to longer-chain PUFA(where the rate of conversion is limiting), they lack the 12- and15-desaturase activities necessary to synthesize the precursor (parent)PUFA, LA and ALA (Knutzon et al., J. Biol. Chem. 273:29360–29366, 1998).Furthermore, the n-3 and n-6 PUFA are not interconvertible in manmaliancells (Goodnight et al., Blood 58: 880–885, 1981). Thus, both LA and ALAand their elongation, desaturation products are considered essentialfatty acids in the human diet. The PUFA composition of mammalian cellmembranes is, to a great extent, dependent on dietary intake (Clandininet al., Can. J. Physiol. Pharmacol. 63:546–556, 1985; McLennan et al.,Am. Heart J. 116:709–717, 1988).

To the contrary, some plants and microorganisms are able to synthesizen-3 fatty acids such as ALA (18:3n-3) because they have membrane-bound12- and 15-(n-3) desaturases that act on glycerolipid substrates in boththe plastid and endoplasmic reticulum (Browse and Somerville, Annu. Rev.Plant Physiol. Plant Mol. Biol. 42: 467–506, 1991). Genetic techniqueshave led to the identification of the genes encoding the 12- and15-desaturases from Arabidopsis thaliana and other higher plant species(Okuley et al., Plant Cell 6:147–158, 1994; Arondel et al., Science258:1353–1355, 1992). Recently, a fat-1 gene encoding an n-3 fatty aciddesaturase was cloned from Caenorhabditis elegans (Spychalla et al.,Proc. Natl. Acad. Sci. USA 94:1142–1147, 1997; see also U.S. Pat. No.6,194,167).

SUMMARY

The present invention is based, in part, on the discovery that the C.elegans n-3 desaturase gene, fat-1, can be successfully introduced intoother types of animal cells (e.g., mammalian cells), where it quicklyand effectively elevates the cellular n-3 PUFA content and dramaticallybalances the ratio of n-6:n-3 PUFAs. More specifically, heterologousexpression of the fat-1 gene in rat cardiac myocytes rendered thosecells capable of converting various n-6 PUFAs to the corresponding n-3PUFA and changed the n-6:n-3 ratio from about 15:1 (an undesirableratio) to 1:1 (a desirable ratio). In addition, an eicosanoid derivedfrom n-6 PUFA (i.e. arachidonic acid) was significantly reduced in thetrasgenic cells (as described further below, levels of arachidonic acidcan be assessed to determine whether a given nucleic acid encodes abiologically active desaturase; similarly, one can assess the levels ofn-6 PUFA; the levels of n-3 PUFA; and the ratio of n-6:n-3 PUFAs).Accordingly, the present invention features compositions (e.g., nucleicacids encoding fat-1, optionally and operably linked to a constitutivelyactive or tissue-specific promoter) and methods that can be used toeffectively modify the content of PUFAs in animal cells (i.e., cellsother than those of C. elegans, for example, mammalian cells such asmyocytes, neurons (whether of the peripheral or central nervous system),adipocytes, endothelial cells, and cancer cells). More generally, afat-1 sequence or a biologically active variant thereof can be operablylinked to a regulatory sequence. Regulatory sequences encompass not onlypromoters, but also enhancers or other expression control sequence, suchas a polyadenylation signal, that facilitates expression of the nucleicacid. The modified cells (whether in vivo or ex vivo (e.g., in tissueculture)), transgenic animals containing them, and food productsobtained from those animals (e.g., meat or other edible parts of theanimals (e.g., liver, kidney, or sweetbreads)) are also within the scopeof the present invention.

In one embodiment, the invention features mammalian cells that contain anucleic acid sequence encoding the C. elegans n-3 desaturase orbiologically active variants (e.g., fragments or other mutants) thereof.Biologically active variants of the n-3 desaturase enzyme are variantsthat retain enough of the biological activity of a wild-type n-3desaturase to be therapeutically or clinically effective (i.e., variantsthat are useful in treating patients, producing transgenic animals, orconducting diagnostic or other laboratory tests). For example, variantsof n-3 desaturase can be mutants or fragments of that enzyme that retainat least 25% of the biological activity of wild-type n-3 desaturase. Forexample, a fragment of an n-3 desaturase enzyme is a biologically activevariant of the full-length enzyme when the fragment converts n-6 fattyacids to n-3 fatty acids at least 25% as efficiency as the wild-typeenzyme does so under the same conditions (e.g., 30, 40, 50, 75, 80, 90,95, or 99% as efficient as wild-type n-3 desaturase). Variants may alsocontain one or more amino acid substitutions (e.g., 1%, 5%, 10%, 20%,25% or more of the amino acid residues in the wild-type enzyme sequencecan be replaced with another amino acid residue). These substitutionscan constitute conservative amino acid substitutions, which are wellknown in the art. Cells that express a fat-1sequence (optionally,operably linked to a constitutively active or tissue-specific promoter)are valuable aids to research because they provide a convenient systemfor characterizing the functional properties of the fat-1 gene and itsproduct (cells in tissue culture are particularly convenient, but theinvention is not so limited). They also allow one to study any cellularmechanism mediated by n-3 fatty acids without the lengthy feedingprocedures of cells or animals that are currently required, and theyserve as model systems that can be used, for example, to evaluateexisting methods and to design new methods for effectively transferringsequences encoding an n-3 desaturase into cells in vivo. In any of thesecontexts (e.g. whether the compositions of the invention are being usedto treat patients, to generate transgenic animals, or in cell cultureassays), nucleic acids encoding fat-1 or a biologically active variantthereof can be co-expressed (by way of the same or a separate vector)with a heterologous gene. The heterologous gene can be, for example,another therapeutic gene (e.g., a receptor for a small molecule orchemotherapeutic agent) or a marker gene (e.g., a sequence encoding afluorescent protein, such as green fluorescent protein (GFP) or enhanced(EGFP)).

The nucleic acids of the invention can be formulated for administrationto a patient. For example, they can be suspended in sterile water or aphysiological buffer (e.g., phosphate-buffered saline) for oral orparenteral administration to a patient (e.g., intravenous,intramuscular, intradermal, or subcutaneous injection (in the event thepatient has a tumor, the compositions can be injected into the tumor oradminstered to the tissue surrounding the site from which a tumor wasremoved) or by inhalation).

The invention also features transgenic animals (including any animalkept as livestock or as a food source) that express the C. elegans n-3desaturase gene or a biologically active variant thereof. Given thediscovery that a C. elegans fat-1 gene can be efficiently expressed whendelivered to a mammalian cell, this gene can be used to generatetransgenic mice or larger transgenic animals (such as cows, pigs, sheep,goats, rabbits or any other livestock or domesticated animal) accordingto methods well known in the art. Depending on whether the constructused contains a constitutively active promoter or a tissue-specificpromoter (e.g., a promoter that is active in skeletal muscle, breasttissue, the colon, neurons, retinal cells, pancreatic cells (e.g., isletcells) etc.) the fat-1 gene can be expressed globally or in atissue-specific manner. The cells of the transgenic animals will containan altered PUFA content that, as described further below, is moredesirable for consumption. Thus, transgenic livestock (or any animalthat is sacrificed for food) that express the desaturase enzyme encodedby the fat-1 gene will be superior (i.e., healthier) sources of food.Food obtained from these animals can be provided to healthy individualsor to those suffering from one or more of the conditions describedbelow.

As noted, the invention features methods of treating patients (includinghumans and other mammals) who have a condition associated with aninsufficiency of n-3 PUFA or an imbalance in the ratio of n-3:n-6 PUFAsby administering a nucleic acid encoding an n-3 desaturase or abiologically active variant thereof (e.g., a fragment or other mutant).Alternatively, one can administer the protein encoded. The methods canbe carried out with patients who have an arrhythmia or cardiovasculardisease (as evidenced, for example, by high plasma triglyceride levelsor hypertension), cancer (e.g., breast cancer or colon cancer),inflammatory or autoimmune diseases (such as rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease (IBD), asthma, chronicobstructive pulmonary disease, lupus, diabetes, Sjogren's syndrometransplantation, ankylosing spondylitis, polyarteritis nodosa, reiter'ssyndrome, and scleroderma), a malformation (or threatened malformation,as occurs in premature infants) of the retina and brain, diabetes,obesity, skin disorders, renal disease, ulcerative colitis, Crohn'sdisease, chronic obstructive pulmonary disease, or who are at risk ofrejecting a transplanted organ. Given that fat-1 expression can alsoinhibit cell death (by apoptosis) in neurons, the methods of theinvention can also be used to treat or prevent (e.g., inhibit thelikelihood of, or the severity of) neurodegenerative diseases.Accordingly, the invention features methods of treating a patient whohas (or who may develop) a neurodegenerative disease such as Parkinson'sdisease, Alzheimer's disease, Huntington's disease (HD), spinal andbulbar muscular atrophy (SBMA; also known as Kennedy's disease),dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1(SCA1), SCA2, SCA6, SCA7, or Machado-Joseph disease (MJD/SCA3) (Reddy etal. Trends Neurosc. 22:248–255, 1999). As a balanced n-6:n-3 ratio isessential for normal growth and development, and as noted above, themethods of the invention can be advantageously applied to patients whohave no discernable disease or condition.

Abbreviations used herein include the following: AA for arachidonic acid(20:4n-6); DHA for docosahexaenoic acid (22:6n-3); EPA foreicosapentaenoic acid (20:5n-3); GFP for green fluorescent protein;Ad.GFP for adenovirus carrying GFP gene; Ad.GFP.fat-1 for adenoviruscarrying both fat-1 gene and GFP gene; and PUFAs for polyunsaturatedfatty acids.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, useful methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflicting subject matter, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a collection of four photomicrographs showing gene transferefficiency. Rat cardiac myocytes were infected with Ad.GFP (left panels;control) or Ad.GFP.fat-1 (right panels). Forty-eight hours afterinfection, cardiomyocytes were visualized with bright light (upperpanels) and at 510 nm of blue light (lower panels). Coexpression of GFPdemonstrates visually that the transgene is being expressed in cellswith a high efficiency.

FIG. 2 is an autoradiogram of a ribonuclease (RNase) protection assay offat-1 transcript levels in cardiac myocytes infected with Ad.GFP(control) and myocytes infected with Ad.GFP.fat-1. Total RNA (10 μg)isolated from the cardiomyocytes was hybridized with anti-sense RNAprobes, digested with RNase and resolved by electrophoresis through adenaturing polyacrylamide gel. The fat-1 mRNA was visualized byautoradiography. A probe targeting β-actin gene was used as control.

FIG. 3. is a pair of partial gas chromatograph traces showing fatty acidprofiles of total cellular lipids extracted from control cardiomyocytesinfected with Ad.GFP and cardiomyocytes infected with Ad.GFP.fat-1.

FIG. 4 is a bar graph depicting prostaglandin E₂ levels in controlcardiomyocytes and cardiomyocytes expressing the fat-1 gene (asdetermined by enzyme immunoassay). Values are means ±SDs of threeexperiments and are expressed as % of control. *p<0.01.

FIG. 5 is a Table showing the polyunsaturated fatty acid composition oftotal cellular lipids from control cardiomyocytes and the transgeniccardiomyocytes expressing a C. elegans fat-1 cDNA.

FIG. 6 is a flowchart of an experimental protocol.

FIG. 7 is a flowchart of an experimental protocol.

FIG. 8 is a flowchart of an experimental protocol.

FIG. 9 is a pair of partial gas chromatograph traces showing fatty acidprofiles of total cellular lipids extracted from control neurons andneurons infected with Ad-GFP-fat-1.

FIG. 10 is a Table comparing the PUFA composition of total cellularlipids from rat cortical neurons (control) and transgenic cellsexpressing a C. elegans fat-1 cDNA (fat-1).

FIG. 11 is a bar graph showing the results of an enzyme immunoassay ofprostaglandin E₂ levels in control neurons and neurons expressing thefat-1 gene. Ad-GFP-fat-1 infected neurons have lower levels of PGE₂relative to control. Values are means±SD of three experiments andexpressed as a percentage of control. *P<0.01.

FIG. 12 is a bar graph representing the results of an MTT assay of cellviability in control and fat-1 expressing cultures. After 24 hours ofgrowth factor withdrawal, the cell viability of neurons expressing thefat-1 gene is 50% higher than control cells (p<0.01).

FIG. 13 is a pair of tracings showing differential responses of myocytesinfected with Ad.GFP and myocytes infected with Ad.GFP.fat-1 to 7.5 mMextracellular calcium.

FIG. 14 is a line graph showing tumor volume over time (0–4 weeks afterviral injection) and thus, the effect of gene transfer on tumor growth.Breast cancer cells (MDA-MB-231) were implanted subcutaneously on theback of nude mice. Three weeks later, the mice were treated withAd.GFP-fat-1 or Ad.GFP (control; 50 μl, 10¹² VP/m) by intratumoralinjection.

FIG. 15 is a table showing PUFA compositions of total cellular lipidsfrom control MCF-7 cells and the transgenic MCF-7 cells expressing a C.elegans fat-1 cDNA.

FIG. 16 is a bar graph depicting the results of an enzyme immunoassay ofprostaglandin E₂ levels in control MCF-7 cells and MCF-7 cellsexpressing fat-1 gene. Values are means±SE of three experiments andexpressed as a percentage of control. (*P<0.05).

FIGS. 17A and 17B are representations of the nucleotide sequence of theC. elegans fat-1 cDNA SEQ ID NO:3 and the deduced amino acid sequence ofthe Fat-1 polypeptide (SEQ ID NO: 4).

DETAILED DESCRIPTION

The studies described below demonstrate that, inter alia, a nucleic acidmolecule encoding an n-3 desaturase can be efficiently expressed in avariety of mammalian cell types and, as a consequence, those cellsproduce significant amounts of n-3 PUFA from endogenous n-6 PUFA andhave a more balanced ratio of n-6 to n-3 PUFA (1:1). The studies werecarried out using recombinant adenoviral expression vectors, which canmediate gene transfer in vivo or in vitro. Adenoviral vectors expressingfat-1, or biologically active variants thereof, as well as other typesof viral and non-viral expression vectors are within the scope of theinvention now claimed. Other viral vectors that can be employed asexpression constructs in the present invention include vectors derivedfrom viruses such as vaccinia virus (e.g., a pox virus or a modifiedvaccinia virus ankara (MVA)), an adeno-associated virus (AAV), or aherpes viruses. These viruses offer several attractive features forvarious mammalian cells. For example, herpes simplex viruses (e.g.,HSV-1) can be selected to deliver fat-1 or a biologically active variantthereof, to neuronal cells (and thereby treat patients withneurodegenerative conditions).

Other retroviruses, liposomes, and plasmid vectors are also well knownin the art and can also be used (e.g., the expression vector pUR278 canbe used when one wishes to fuse a fat-1 sequence to the lacZ gene; lacZencodes the detectable marker β-galactosidase (see, e.g., Ruther et al.,EMBO J., 2:1791, 1983). A fat-1 sequence can also be fused to othertypes of heterologous sequences, such as a sequence that encodes anothertherapeutic gene or a sequence that, when expressed, improves thequantity or quality (e.g., solubility or circulating half-life) of thefusion protein. For example, pGEX vectors can be used to express theproteins of the invention fused to glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can be easily purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors (PharmaciaBiotech Inc; Smith and Johnson, Gene 67:31–40, 1988) are designed toinclude thrombin or factor Xa protease cleavage sites so that the clonedtarget gene product can be released from the GST moiety. Other fusionpartners include albumin and a region (e.g., the Fc region) of animmunoglobulin molecule (e.g., IgG, IgA, IgM, or IgE). Other usefulvectors include pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.), which fuse maltose E binding protein andprotein A, respectively, to an n-3 desaturase.

Transgene expression can be sufficiently prolonged from episomalsystems, so that readministration of the vector, with its transgene, isnot necessary. Alternatively, the vector can be designed to promoteintegration into the host genome, preferably in a site-specificlocation, which would help ensure that the transgene is not lost duringthe cell's lifetime. Whatever the means of delivery, transcriptionalcontrol, exerted by the host cell, would promote tissue specificity andregulate transgene expression.

The expression vector will be selected or designed depending on, forexample, the type of host cell to be transformed and the level ofprotein expression desired. For example, when the host cells aremammalian cells, the expression vector can include viral regulatoryelements, such as promoters derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. The nucleic acid inserted (i.e.,the sequence to be expressed; here, fat-1) can also be modified toencode residues that are preferentially utilized in E. coli (Wada etal., Nucleic Acids Res. 20:2111–2118, 1992). These modifications can beachieved by standard recombinant techniques. More generally, theexpression vectors of the invention can be designed to express proteinsin prokaryotic or eukaryotic cells. For example, polypeptides of theinvention can be expressed in bacterial cells (e.g., E. coli), fungi,yeast, or insect cells (e.g., using baculovirus expression vectors). Forexample, a baculovirus such as Autographa californica nuclearpolyhedrosis virus (AcNPV), which grows in Spodoptera frugiperda cells,can be used as a vector to express foreign genes.

As noted elsewhere, the expression vectors and nucleic acids used toexpress fat-1 can also contain a tissue-specific promoter. Suchpromoters are known in the art and include, but are not limited toliver-specific promoters (e.g., albumin; Miyatake et al., 1997),muscle-specific promoters (e.g., myosin light chain 1 (Shi et al., 1997)α-actin), pancreatic-specific promoter (e.g., insulin or glucagonpromoters), neural-specific promoters (e.g., the tyrosine hydroxylasepromoter or the neuron-specific enolase promoter), endothelialcell-specific promoters (e.g., von Willebrandt; Ozaki et al., 1996), andsmooth muscle-cells specific promoters (e.g., 22a; Kim et al., 1997).Tumor-specific promoters are also being used in developing cancertherapies, including tyrosine kinase-specific promoters for B16 melanoma(Diaz et al., 1998), DF3/MUC1 for certain breast cancers (Wen et al.,1993; for breast cancer, an adipose-specific promoter region of humanaromatase cytochrome p450 (p450arom) can also be used (see U.S. Pat. No.5,446,143; Mahendroo et al., J. Biol. Chem. 268:19463–19470, 1993; andSimpson et al., Clin. Chem. 39:317–324, 1993). An α-fetoprotein promotercan be used to direct expression in hepatomas (Chen et al., 1995). Thevectors and other nucleic acid molecules of the invention (e.g., thefat-1 cDNA per se) can also include sequences that limit the temporalexpression of the transgene. For example, the transgene can becontrolled by drug inducible promoters by, for example including cAMPresponse element enhancers in a promoter and treating the transfected orinfected cell with a cAMP modulating drug (Suzuki et al., 1996).Alternatively, repressor elements can prevent transcription in thepresence of the drug (Hu et al., 1997). Spatial control of expressionhas also been achieved by using ionising radiation (radiotherapy) inconjunction with the erg1 gene promoter (Hallaham et al., 1995).Constructs that contain such regulatory sequences are within the scopeof the present invention.

In the examples that follow, RNA analysis and enzymatic assays wereperformed to assess gene expression, and gas chromatography-massspectrometry were used to determine fatty acid profiles (these arestandard techniques that one of ordinary skill in the art could use toassess any variant of the fat-1 sequence for biological activity; orincorporate in any method of assessing a sample obtained from a patientfor fat-1 expression).

Some of the studies described below were conducted using corticalneurons. Fat-1 expression not only modified the cellular n-6:n-3 fattyacid ratio and eicosanoid profile in these neurons, but also protectedthe cells from apoptosis, thereby increasing cellular viability. Morespecifically, fat-1expression modified the fatty acid ratio andprotected rat cortical neurons against growth factor withdrawal-inducedapoptotis in the absence of supplementation with exogenous n-3 PUFAs.Accordingly, the nucleic acid molecules (and other compositions)described herein can be used as neuroprotectants, which can beadministered to premature infants and to older patients having anyneurodegenerative disease (alternatively, the molecules or othercompositions can be delivered to an animal, parts of which are thenconsumed by the patient). The protective effect of gene transfer onneuronal apoptosis minics the protective effects of n-3 fatty acidsupplementation.

The positive results obtained with neurons are especially encouragingbecause n-3 PUFA deficiency leads to abnormal development of the retinaand the brain, particularly in premature infants (Uauy et al., Lipids36:885–895, 2001), and animals deficient in n-3 PUFA show deficits inmemory, spatial and context-dependent learning, and loss of visualacuity (Carrie et al., Neurosci. Lett. 266:69–72, 1999; Yehuda et al.,J. Neurosci. Res. 56:565–70, 1999). There are also indications thatvarious neurological disease states in humans are associated with an n-3deficient status (Vancassel et al., Prost. Leuk. Ess. Fatt. Acids65:1–7, 2001; Hoffman and Birch, World Rev. Nutr. Diet 83:52–69, 1998).

The biological functions of PUFAs are described further here, as thesefunctions bear on the types of conditions amenable to treatment with thenucleic acid molecules (and other compositions) described herein. PUFAsare important structural components of membrane phospholipids and areprecursors of families of signaling molecules (eicosanoids) includingprostaglandins, thromboxanes, and leukotrienes (Needleman et al., Ann.Rev. Biochem. 55:69–102, 1986; Smith and Borgeat, In Biochemistry ofLipids and Membranes, D. E. Vance & J. E. Vance, Eds.,Benjamin/Cummings, Menlo Park, Calif., 00 325–360, 1986). Theeicosanoids derived from PUFAs play a key role in modulatinginflammation, cytokine release, the immune response, plateletaggregation, vascular reactivity, thrombosis and allergic phenomena(Dyerberg et al., Lancet 2:117–119, 1978; Cyerberg and Bang, Lancet2:433–435, 1979; James et al., Am. J. Clin Nutr. 7:343S-3438S, 2000;Calder, Ann. Nutr. Metab. 41:203–234, 1997). The principal fatty acidprecursors of these signaling compounds are arachidonic acid (AA,20:4n6), providing an n-6 substrate that is responsible for the majorsynthesis of the series 2 compounds, and eicosapentaenoic acid (EPA,20:5n3), an n-3 substrate that is responsible for the parallel synthesisof many series 3 eicosanoids with an additional double bond. The n-6:n-3ratio in phospholipids modulates the balance between eicosaniods of the2 and 3 series derived from AA and EPA. The eicosanoids derived from AA(series 2) and EPA (series 3) are functionally distinct and some haveimportant opposing physiological functions (Dyerberg et al., Lancet2:117–119, 1978; Cyerberg and Bang, Lancet 2:433435, 1979; James et al.,Am. J. Clin Nutr. 7:343S–3438S, 2000; Calder, Ann. Nutr. Metab.41:203–234, 1997). Series 3 eicosanoids are weak agonists or, in somecases, antagonists of series 2 eicosanoids. For example, eicosanoids ofthe 2 series promote inflammation and platelet aggregation, and activatethe immune resoponse, whereas series 3 eicosanoids tend to amelioratethese effects. In addition, PUFAs, in the form of free fatty acids, areinvolved in gene expression and intercellular cell-to-cell communication(Price et al., Curr. Opin. Lipidol 11:3–7,2000; Sellmayer et al. Lipids31 Suppl:S37–S40, 1996; vonSchacky, J. Lab. Clin. Med. 128:5–6, 1996).Thus, PUFA can exhibit many diverse biological effects.

The compositions and methods described herein can be used to treat avariety of specific conditions as well as to improve general health. Anycondition that is amenable to treatment by administration of n-3 PUFAsis amenable to treatment by way of the methods of the present invention,which comprise administration of a gene encoding an n-3 desaturase(e.g., the C. elegans fat-1 gene). Some of the conditions amenable totreatment are described below.

n-3 PUFAs have attracted considerable interest as pharmaceutical andnutraceutical compounds (Connor, Am. J. Clin. Nutr. 70:560S–569S, 1999;Simopoulos, Am. J. Clin. Nutr. 70:562S–569S, 1999; Salem et al., Lipids31:S1-S326, 1996). During the past 25 years, more than 4,500 studieshave explored the effects of n-3 fatty acids on human metabolism andhealth (e.g., cardiovascular health). From epidemiology to cell cultureand animal studies to randomized controlled trials, the cardioprotectiveeffects of omega-3 fatty acids have been recognized (Leaf and Kang,World Rev. Nutr. Diet. 83:24–37, 1998; De Caterina et al., Eds., n-3Fatty Acids and Vascular Disease, Springer-Verlag, London, 1999, pp 166;O'Keefe and Harris, Mayo Clin. Proc. 75:607–614, 2000). The predominantbeneficial effects include a reduction in sudden death (Albert et al.,JAMA 279:23–28, 1998; Siscovick et al., JAMA 274:1363–1367, 1995),decreased risk of arrhythmia (Kang and Leaf, Circulation 94:1774–1780,1996), lower plasma triglyceride levels (Harris, Am. J. Clin. Nutr.65:1645S–1654S, 1997), and a reduced blood-clotting tendency (Agren etal., Prostagland. Leukot. Esseizt. Fatty Acids 57:419–421, 1997; Mori etal., Arterioscler. Throm. Basc. Biol. 17:279–286, 1997). Evidence fromepidemiological studies shows that another n-3 fatty acid, α-linolenicacid, reduces risk of myocardial infarction (Guallar et al.,Arterioscler. Thromb. Vasc. BioL 19:1111–1118, 1999) and fatal ischemicheart disease in women (Hu et al., Am. J. Clin. Nutr. 69:890–897, 1999).Several randomized controlled trials recently have demonstratedbeneficial effects of both α-linolenic acid (de Lorgeril et al.,Circulation 99:779–785, 1999) and marine ω-3 fatty acids (Singh et al.,Cardiovasc. drugs ther. 11:485–491, 1997; Von Schacky et al., Ann.Intern. Med. 130:554–562, 1999; GISSI-Prevenzione Investigators, Lancet354:447–455, 1999) on both coronary morbidity and mortality in patientswith coronary disease. The n-3 fatty acid, EPA, exerts anticanceractivity in vitro and in animal models of experimental cancer (Bougnoux,Curr. Opin. Clin. Nutr. Metab. Care 2:121–126, 1999; Cave, Breast CancerRes. Treat. 46:239–246, 1997). Human studies show that populations whosediets are rich in EPA exhibit a remarkably low incidence of cancer (Roseand Connolly, Pharmacol. Ther. 83:217–244, 1999). Supplementation withn-3 PUFAs shows therapeutic effects on inflammatory and autoimmunediseases such as arthritis (Kremer, Am. J. Clin. Nutr. 71:349S–351 S,2000; Ariza-Ariza et al., Semin. Arthritis Rheum. 27:366–370, 1998;James et al., Am. J. Clin. Nutr. 71:343S–348S), and studies withnonhuman primates (Neuringer et al., Proc. Natl. Acad. Sci. USA83:4021–4025, 1986) and human newborns (Uauy et al., Proc. Nutr. Soc.59:3–15, 2000; Uauy et al., Lipids 31:S167–176, 1996) indicate that then-3 fatty acid, DHA, is essential for the normal fimctional developmentof the retina and brain, particularly in premature infants. Furthermore,n-3 PUFA have been shown to have beneficial effects on many otherclinical problems, such as hypertension (Appel et al., Arch. Intern.Med. 153:1429–1438, 1993), diabetes (Raheja et al., Ann. N.Y. Acad. Sci.683:258–271, 1993), obesity (Clarke, Br. J. Nutr. 83:S59–66, 2000), skindisorders (Ziboh, World Rev. Nutr. Diet. 66:425–435, 1991), renaldisease (De Caterina et al., Kidney Int. 44:843–850, 1993), ulcerativecolitis (Stenson et al., Ann. Intern. Med. 116:609–614, 1992), Crohn'sdisease (Belluzzi et al., N. Engl. J. Med. 334:1557–1560, 1996), chronicobstructive pulmonary disease (Shahar et al., N. Engl. J. Med.331:228–233, 1994), and transplanted organ rejection (Otto et al.,Transplantation 50:193–198, 1990). In general, a balanced n-6:n-3 ratioof the body lipids is essential for normal growth and development andplays an important role in the prevention and treatment of many clinicalproblems. The diseases, disorders, and conditions described above areamenable to treatment with the nucleic acid molecules (and othercompositions) described herein.

According to recent studies (Simopoulos, Poultry Science 79:961–970,2000), the ratio of n-6 to n-3 essential fatty acids in today's diet isaround 10–20:1. This indicates that present Western diets are deficientin n-3 fatty acids compared with the diet on which humans evolved andtheir genetic patterns were established (n-6/n-3=1:1) (Leaf and Weber,Am. J. Clin Nutr. 45:1048–1053, 1987). Since the n-6 and n-3 fatty acidsare metabolically and functionally distinct and have important opposingphysiological functions, their balance is important for homeostasis andnormal development. However, n-3 and n-6 PUFAs are not interconvertiblein the human body because mammalian cells lack the enzyme n-3desaturase. Therefore, the balance between n-6 and n-3 PUFA inbiological membranes is regulated based on dietary supply. Elevating thetissue concentrations of n-3 fatty acids in human subjects or animalsrelies on increased consumption of n-3 PUFA-enriched foods or n-3 PUFAsupplements. Given the potential therapeutic actions of n-3 PUFAs, aninternational scientific working group has recommended diets in whichthe intake of n-6 fatty acids is decreased and the intake of n-3 fattyacids is increased (Simopoulos, Food Australia 51:332–333, 1999). TheAmerican Heart Association has also recently made such a dietaryrecommendation (AHA Dietary Guidelines: Revision 2000, Circulation102:2284–2299, 2000).

Although dietary supplementation with n-3 PUFA is a safe intervention,it has a number of limitations. For example, to achieve a significantincrease in tissue concentrations of n-3 PUFA in vivo requires a chronicintake of high doses of n-3 PUFA for a period of at least 2–3 months.Bioavailability of fatty acids to cells from the diet involves a seriesof physiological processes including digestion, absorption, transportand metabolism of fat. Thus, the efficacy of dietary interventiondepends on the physiological and health status of an individual. Apatient in critical condition or who has a gastrointestinal disorder isunlikely to be able to ingest or absorb fatty foods or n-3 PUFAsupplements. In addition, encapsulated fish oil supplements are unlikelyto be suited to daily use over a person's lifetime because of their highcaloric content. Moreover, ingestion of some species of fish from costalwaters and lakes may carry toxic amounts of mercury or organic toxins,and effective dietary intervention requires a disciplined change indietary habits that some people may not be able to sustain. In view ofthe foregoing, there is a great need for the means to quickly andeffectively increase cellular n-3 PUFA content and balance the n-6:n-3ratio without resorting to long-term intake of fish or fish oilsupplements. This need is met by the methods of the present invention,which create an alternative food source (via transgenic livestock whosecells contain substantially more n-3 PUFAs than in non-transgenicanimals) or which provide for administration of a gene encoding an n-3desaturase enzyme to patients (e.g., human patients). A particularadvantage of the present methods is that they not only elevate tissueconcentrations of n-3 PUFAs, but also simultaneously decreases thelevels of excessive endogenous n-6 PUFA.

EXAMPLES Example 1 Construction of a Recombinant Adenovirus

A recombinant adenovirus carrying the fat-1 gene was constructedfollowing procedures similar to those described by He et al. (Proc.Natl. Acad. Sci. USA 95:2509–2514, 1998). The n-3 fatty acid desaturasecDNA (fat-1 gene) in pCE8 was kindly provided by Dr. J. Browse(Washington State University) (but can be synthesized or cloned usinginformation and techniques available to those of ordinary skill in theart; see Spychalla et al., Proc. Natl. Acad. Sci. USA 94:1142–1147,1997; U.S. Pat. No. 6,194,167; and FIGS. 17A and 17B). The cDNA insertof pCE8 was excised from the plasmid with an EcoRI/KpnI double digest,inserted into a shutter vector, and then recombined with an adenoviralbackbone according to the methods of He et al. (supra). Two,first-generation type 5 recombinant adenoviruses were generated: Ad.GFP,which carries the green fluorescent protein (GFP, as reporter gene)under control of the cytomegalvirus (CMV) promoter, and Ad.GFP.fat-1,which carries both the fat-1 and GFP genes, each under the control ofseparate CMV promoters. The recombinant viruses were prepared as hightiter stocks through propagation in 293 cells, as described previously(Hajjar et al. Circulation 95:423–429, 1997). The constructs wereconfirmed by enzymatic digestion and by DNA sequence analysis. See alsoHajjar et al., Circulation 95:4230429, 1997 and Hajjar et al., Circ.Res. 81:145–153, 1997.

Wild-type adenovirus contamination can be assessed and shown to beexcluded by the absence of both PCR-detectable E1 sequences andcytopathic effects on the nonpermissive A549 cell line. Alternativeadenoviral vectors with other promoters or adeno-associated viral (AAV)vectors can be constructed if necessary or desired.

Example 2 Culture and Infection of Cardiac Myocytes with Adenovirus

Cardiac myocytes were isolated from one-day-old rats using the NationalCardiomyocyte Isolation System (Worthington Biochemical Corp., Freehold,N.J.). The isolated cells were placed in 6-well plates and cultured inF-10 medium containing 5% fetal bovine serum and 10% horse serum at 37°C. in a tissue culture incubator with 5% CO₂ and 98% relative humidity.Cells were used for experiments after 2–3 days of culture. Viralinfection was carried out by adding viral particles at differentconcentrations (5×10^(9–10) ¹⁰ pfu) to culture medium containing 2%fetal bovine serum (FBS). After a 24 hour incubation, the infectionmedium was replaced with normal (15% serum), culture medium supplementedwith 10 μM of 18:2n-6 and 20:4n-6. About 48 hours after infection, thecells can be used (e.g., one can then analyze gene expression, fattyacid composition, viability, or growth (e.g., proliferation or rate ofdivision)).

Example 3 Detecting Fat-1 Expression with Fluorescence Microscopy andRNA Analysis

Gene expression can be assessed by many methods known in the art ofmolecular biology. Here, expression of fat-1 in cardiac myocytes,infected as described above, was assessed by visual examination ofinfected cells and a ribonuclease (RNase) protection assay.

More specifically, the coexpression of GFP allowed us to identify thecells that were infected and expressed the transgene. About 48 hoursafter infection, almost all of the cells (>90%) exhibited brightfluorescence, indicating a high efficiency of gene transfer and a highexpression level of the transgene (see FIG. 1). Expression of fat-1transcripts was also determined by RNase protection assay using a RPAIII™ it (Ambion). Briefly, total RNA was extracted from cultured cellsusing an RNA isolation kit (Qiagen) according to the manufacturer'sprotocol. The plasmid containing the fat-1 gene, pCE8, was linearizedand used as a transcription template. Anti-sense RNA probes weretranscribed in vitro using ³³P-UTP, hybridized with the total RNAextracted from the myocytes, and digested with RNase to removenon-hybridized RNA and probe. The protected RNA:RNA was resolved byelectrophoresis through a denaturing gel and subjected toautoradiography. A probe targeting the β-actin gene was used as acontrol. Fat-1 mRNA was not detected in cells infected with AD.GFP (alsoused as a control), but was abundant in cells infected with Ad.GFP.fat-1(FIG. 2). This result indicates that adenovirus-mediated gene transferconfers very high expression of fat-1 gene in rat cardiac myocytes thatnormally lack the gene.

Example 4 Lipid Analysis; The Effect of n-3 Sesaturase on Fatty AcidComposition

By lipid analysis, one can determine whether the expression of a fat-1gene in cardiac myocytes (or any other cell type) converts n-6 fattyacids to n-3 fatty acids and, thereby, changes the fatty acidcomposition of the cell. Following infection with the adenovirusesdescribed above, cells were incubated in medium supplemented with n-6fatty acids (10 μM 18:2n-6 and 10 μM 20:4n-6) for 2–3 days. After theincubation, the fatty acid composition of total cellular lipids wasanalyzed as described previously (Kang et al., Biochim. Biophys. Acta.1128:267–274, 1992; Weylandt et al., Lipids 31:977–982, 1996).

Lipid was extracted with chloroform/methanol (2:1, v/v) containing0.005% butylated hydroxytoluene (as antioxidant). Fatty acid methylesters were prepared using 14% BF3/methanol reagent. Fatty acid methylesters are quantified by GC/MS using a HP5890 Series II gaschromatograph equipped with a Supelcowax SP-10 capillary column attachedto a HP-5971 mass spectrometer. The injector and detector are maintainedat 260° C. and 280° C., respectively. The oven program is initiallymaintained at 150° C. for 2 minutes, then ramped to 200° C. at 10°C./min and held for 4 minutes, ramped again at 5° C./min to 240° C.,held for 3 minutes, and finally ramped to 270° C. at 10° C./min andmaintained for 5 minutes. Carrier gas flow rate is maintained at aconstant 0.8 mL/min throughout. Total ion monitoring is performed,encompassing mass ranges from 50–550 amus. Fatty acid mass is determinedby comparing areas of various analyzed fatty acids to that of a fixedconcentration of internal standard.

The fatty acid profiles were remarkably different between the controlcells infected with Ad.GFP and the cells infected with Ad.GFP.fat-1(FIG. 3). Moreover, cells infected with Ad.GFP showed no change in theirfatty acid profiles when compared with non-infected cells. In the cellsexpressing the fat-1 gene (n-3 desaturase), almost all kinds of n-6fatty acids were largely converted to the corresponding n-3 fatty acids,namely, 18:2n-6 to 18:3n-3, 20:2n-6 to 20:3n-3, 20:3n-6 to 20:4:n-3,20:4n-6 to 20:5n-3, and 22:4n-6 to 22:5n-3. As a result, the fatty acidcomposition of the cells expressing fat-1 was significantly changed withrespect to that of the control cells infected with Ad.GFP (FIG. 5).Importantly, the ratio of n-6:n-3 was reduced from 15:1 in the controlcells to 1:1.2 in the cells expressing the n-3 fatty acid desaturase.

Example 5 Measuring Eicosanoids Following Fat-1 Expression

Since 20:4n-6 (AA) and 20:5n-3 (EPA) are the precursors of 2-series and3-series of eicosanoids, respectively, differences in the contents of AAand EPA may lead to a difference in production of eicosanoids in thecells. Thus, we measured the production of eicosanoids in the infectedcells following stimulation with calcium ionophore A23187 by using a EIAkit that specifically detect prostaglandin E₂ with a 16%cross-reactivity with prostaglandin E3. More specifically, ProstaglandinE₂ was measured by using enzyme immunoassay kits (Assay Designs, Inc)following the manufacturer's protocol. (The cross-reactivity with PGE3is 16%). Cultured cells were washed and serum-free medium containingcalcium ionophore A23187 (5 μM). After a 10 minute incubation, theconditioned medium was recovered and subjected to eicosanoidmeasurement. The amount of prostaglandin E₂ produced by the controlcells was significantly higher than that produced by cells expressingthe n-3 desaturase encoded by fat-1 (FIG. 4).

Example 6 Analysis of Animal Cells in Culture

In this example and the two that follow, we set out three differentexperimental models: cultured cells (other types of cultured cells aretested further below), adult rats, and transgenic mice. As shown above,the cultured cell model can be used to characterize the enzymaticproperties and biochemical effects of the n-3 desaturase when expressedin mammalian cells in vitro; the adult rat model can be used to evaluatethe efficacy with which a transferred fat-1 gene can elevate tissueconcentrations of n-3 PUFA in vivo, and the transgenic mouse model canbe used to assess the long-term and systematic effects of the transgeneon lipid composition of various tissues or organs in vivo. For the firsttwo models, the introduction of the fat-1 gene into mammaliancells/tissues will be carried out by mean of adenoviral gene transfer(mediated by recombinant adenoviruses). For the last model, genetransfer will be carried out by microinjection of the transgene intofertilized mouse eggs. Following gene transfer, the expression profileof the transferred gene can be characterized by mRNA and/or proteinanalysis (see, e.g., Example 3, above), and the biochemical effects,mainly the fatty acid composition of the cells or tissues, will bedetermined by GC-MS technology (see, e.g., Example 4, above).Eicosanoids will be measured by enzyme immunoassay (see, e.g., Example5). Changes are identified by comparing the data obtained fromfat-1-expressing cells with data obtained from control cells or tissuesinfected with the same (or a similar) virus, but not transfected withfat-1. The end point of these studies is the biochemical changes incellular fatty acid composition and eicosanoid profile.

Cultures of virtually any animal cells (including human cell lines) canbe infected with recombinant adenovirus (Ad.GFP.fat-1 or Ad.GFP), afterwhich expression of the transferred gene can be assessed by RNA orprotein analysis. The experimental procedures and related methods aredescribed in the Examples above and outlined in FIG. 6. Various celltypes including cardiac myocytes, neurons, hepatocytes, endothelialcells, and macrophages have been used in studies of n-3 fatty acids.

Cardiac myocytes can be isolated and cultured as described above (seeExample 2), and other cell types, such as cerebellar granule neurons andhepatocytes can be prepared from 1–5 day-old rats following the methoddescribed by Schousboe et al. (In A Dissection and Tissue Culture Manualof the Nervous System, Shahar et al., Eds., Alan R. Liss, New York,N.Y., pp. 203–206, 1989). Human cell lines, including breast cancer celllines and leukemia cell lines can be cultured in MEN medium or RPMI 1640supplemented with 10% fetal bovine serum (FBS) in a 37° C./5% CO₂incubator.

Viral infection can be carried out by adding viral particles at variousconcentrations (e.g., 2×10^(9–2×10) ¹⁰ pfu) to culture medium containingno FBS or 2% FBS (see also Example 2). ARer a 24-hour incubation, theinfection medium is replaced with normal (10% FBS) culture medium.Forty-eight hours after infection, cells can be used for analysis ofgene expression or fatty acid composition. Transgene expression can beassessed by fluorescence microscopy when a fluorescent tag is includedin the transgene (see Example 1 and FIG. 1; similarly, the tag can be anantigenic protein detected by a fluorescent antibody) or by a standardRNA assay (e.g. a Northern blot or RNase protection assay). Since thefat-1 gene normally does not exist in control cells, it is not difficultto identify the difference in fat-1 mRNA between the control cells andcells expressing fat-1.

n-3 desaturase catalyzes the introduction of an n-3 double bond into n-6fatty acids, leading to formation of n-3 fatty acids with one moredouble bond than their precursor n-6 fatty acids (e.g., 18:2n-6→18:3n-3,20:4n-6→20:5n-3). The rate of conversion of substrates to products (theamount of products formed within a given time period) is thought to bedirectly proportional to the expression/activity of a desaturase. Thus,the functional activity of this enzyme can be determined, from a sampleobtained from an animal (e.g., a tissue sample) or in cultured cells bymeasurement of the conversions (the quantity of products) using thefollowing methods.

Fatty acid desaturation assay using radiolabeled n-6fatty acids assubstrates: The assay can be performed following the protocol describedby Kang et al. (Biochim. Biophys Acta. 1128:267–274, 1992). Briefly,various labeled n-6 fatty acids (e.g., [¹⁴C]18:2n-6, [¹⁴C]20:4n-6) boundto BSA are added to serum-free culture medium and incubated with cellsfor 4–6 hours. After that, cells and culture medium will be harvested.Lipids are extracted and methylated (see below). The labeled fatty acidmethyl esters are separated according to degree of unsaturation (i.e.,the number of double bond) on silica-gel TLC plates impregnated withAgNO₃. Bands containing fatty acids with different double bonds can beidentified by comparison with reference standards. Quantity of thelabeled fatty acids is determined by scintillation counting, and dataare compared between control cells and the cells expressing the fat-1gene.

Fatty acid analysis by gas chromatography: Conversion of fatty acids canbe determined more accurately by analysis of fatty acid compositionusing gas chromatography-mass spectrometry (see below). Using thismethod, no radiolabeled fatty acid is required. Fatty acid contents ofcultured cells expressing the n-3 desaturase gene, in the presence ofvarious substrates, can be analyzed. The conversion of each fatty acidcan be determined by comparison of fatty acid profiles between controlcells and the cells expressing the fat-1 gene.

The fatty acid composition of total cellular lipids or phospholipids canbe analyzed as described previously (Kang et al., Biochim. Biophys.Acta. 1128:267–274, 1992; Weylandt et al., Lipids 31:977–982, 1996). Theprocedures are as follows:

Lipid extraction (see also Example 4): Five ml of chloroform/methanol(2:1, v/v) containing 0.005% butylated hydroxytoluene (as antioxidant)is added to washed cell pellets and vortexed vigorously for 1 minutethen left at 4° C. overnight. One ml of 0.88% NaCl is added and mixedagain. The chloroform phase containing lipids is collected. The remainsare extracted once again with 2 ml chloroform. The chloroform is pooledand dried under nitrogen and stored in sealed tubes at −70° C.

Separation of lipids by thin-layer chromatography (TLC): TLC plates areactivated at 100° C. for 60 minutes. TLC tanks are equilibrated withsolvent for at least one hour prior to use. Total phospholipid andtriacyglycerol are separated by running the sample on silica-gel Gplates using a solvent system comprised of petroleum ether/diethylether/acetic acid (80:20:1 by vol.) for 30–35 minutes. Individualphospholipids are separated by TLC on silica-gel H plates using thefollowing solvent system: chloroform/methanol/2-propanol/0.25%KCl/triethylamine (30:9:25:6:18 by vol.). Bands containing lipids aremade visible with 0.01% 8-anilino-1-naphthalenesulfonic acid, and gelscrapings of each lipid fraction are collected for methylation.

Fatty acid methylation: Fatty acid methyl esters are prepared using 14%BF₃/methanol reagent. One or two ml of hexane and 1 ml of BF₃/methanolreagent are added to lipid samples in glass tubes with Teflon-linedcaps. After being flushed with nitrogen, samples are heated at 100° C.for one hour, cooled to room temperature and methyl esters are extractedin the hexane phase following addition of 1 ml H₂O. Samples are allowedto stand for 20–30 minutes, the upper hexane layer is removed andconcentrated under nitrogen for GC analysis.

Gas chromatography-mass spectrometry. Methylated samples arereconstituted in 100–200 μl hexane or isooctane of which 1–2 μl will beanalyzed by gas chromatography. An Omegawas column (30 m; Supelco,Bellefonte, Pa.) will be used in a Hewlett-Packard 5890A gaschromatograph (Hewlett-Packard, Avondage, Pa.). Carrier gas is hydrogen(2.39 ml/min), injected with a split ratio of 1:31. The temperature isinitially 165° C. for 5 minutes, then is increased to 195° C. at 2.5°C./min and, from there, to 220° C. at 5° C./min. The temperature is heldfor 10.5 minutes and then decreased to 165° C. at 27.5° C./min. Peakswill be identified by comparison with fatty acid standards(Nu-Chek-Prep, Elysian, Minn.), and area percentage for all resolvedpeaks will be analyzed using a Perkin-Elmer M1 integrato (Perkin-Elmer,Norwood, Conn.). These analytical conditions separates all saturated,mono, di- and polyunsaturated fatty acids from C14 to C25 carbons inchain length. The sample size will be calculated based on externalstandards when added. In addition, the gas chromatography-massspectrometry (GC-MS) will be carried out using a Hewlett-Packard massselective detector (model 5972) operating at an ionization voltage of 70eV with a scan range of 20–500 Da. The mass spectrum of any new peakobtained will be compared with that of standards (Nu Chek Prep, Elysian,Minn.) in the database NBS75K.L (National Bureau of Standards).

Example 7 Evaluation of n-3 desaturase Gene Transfer In Vivo

The experiments described here allow introduction of the fat-1 gene intoanimal tissues or organs (e.g., heart), where the enzyme product canquickly optimize fatty acid profiles by increasing the content of n-3PUFAs and decreasing the content of n-6 PUFAs. The heart is selected asan experimental target for the gene transfer because it has been wellstudied in relation to n-3 fatty acids, and it is a vital organ.

Adult rats, fed a normal diet or a diet high in n-6 PUFA for two months,will be randomized to receive either an adenovirus carrying the fat-1gene (Ad.GFP.fat-1) or an adenovirus carrying the reporter gene GFP (Ad.GFP, as control). The adenoviruses will be delivered to the heart of aliving animal using a catheter-based technique, which can produce anexpression pattern that is grossly homogeneous throughout the heart(Hajjar et al., Proc. Natl. Acad. Sci. USA 95:525105256, 1998). Twodays, 4 days, 10 days, 30 days and 60 days after infection (genetransfer), animals will be sacrificed, and their hearts will beharvested and used for determination of the transgene expression andanalysis of fatty acid composition. Another group of rats will be fed adiet rich in n-3 fatty acids (low n-6/n-3 ratio) for two months withoutgene transfer and used as references. These experiments (in whichanimals are on different diets and samples harvested at different timepoints) are designed to determine whether transfer of the fat-1 gene canbring about a desired biochemical effect (n-6/n-3 ratio, eicosanoidprofile) similar to or even superior to that induced by dietaryintervention (i.e., n-3 FA supplementation), how quickly a significantchange in fatty acid composition can be achieved, and how long thechange can last. Rats injected with the reporter (GFP) gene will be usedas controls (our preliminary studies showed that gene transfer of GFPhas no effect on fatty acid composition). The experimental flow chart isshown in FIG. 7.

Animals and Diets: weight-matched adult Sprague-Dawley rats will berandomly assigned to three groups. Each group is fed with one of threedifferent diets: normal (basal) diet, a high n-6 diet, or a high n-3diet. These diets are prepared as follows.

Basal diet: a commercial rat fat-free diet (Agway Inc. C.G., Syracuse,N.Y.) to which 2% (w/w) corn oil is added; High n-6 diet: the basal dietsupplemented by addition of a further 13% (w/w) corn oil or saffloweroil (high in n-6 fatty acids), bringing the final diet to a total of 15%fat; High n-3 diet: the basal diet supplemented with 13% (w/w) fish oil(30% EPA, 20% DHA, 65% total n-3 PUFA) (Pronova Biocare A/S, Oslo),bringing the final diet to a total of 15% fat. This group will serve asa control group for this study.

The diets will be prepared in small batches weekly, kept at −20° C. andthawed daily in the amounts required. Vitamin E (100 mg/100 g fat) andbutylated hydroxy toluene (final concentration 0.05%) will be added toprevent oxidation of long-chain polyunsaturated fatty acids (The BHTshould serve to prevent autooxidation of the unsaturated fatty acidsduring preparation and storage). To ensure animals are receivingadequate nutrition, the rats in all groups will be weighted weekly.After 8 weeks on the diets, the animals will be subjected to genetransfer.

Adenoviral Delivery Protocol. The delivery of adenoviruses to the heartwill be performed by using a cathether-based technique similar to thatdescribed by Hajjar et al (supra). Briefly, rats will be anesthetizedwith intra peritoneal pentobarbital (60 mg/kg) and placed on aventilator. The chest is entered from the left side through the thirdintercostals space. The pericardium is opened and a 7–0 suture placed atthe apex of the left ventricle. The aorta and pulmonary artery areidentified. A 22-gauge catheter containing 200 μL adenovirus (9–10×10¹⁰pfu/ml) is advanced from the apex of the left ventricle (LV) to theaortic root. The aorta and pulmonary arteries are clamped distal to thesite of the catheter, and the solution is injected. The clamp ismaintained for 10 seconds while the heart pumped against a closed system(isovolumically). After 10 seconds, the clamp on the aorta and pulmonaryartery is released, the chest is closed, and the animals are extubatedand transferred back to their cages.

At day 2, 4, 10, 30 and 60 after gene transfer, animals will besacrificed, their hearts infected with the viruses will be removed,perfused or rinsed with saline to removed all blood and a portion of thetissues will be promptly frozen at −80° C. for lipid analysis andeicosanoid measurement. The remaining tissues will be used fordetermination of the mRNA levels and/or protein levels of the n-3desaturase.

It is possible that other organs such as brain and liver may also beinfected at high levels by the adenoviruses entering the blood stream.Thus, other organs, in addition to the heart, will be also harvested foranalyses of transgene expression and lipid profile.

Other methods, including assessment of transgene expression (by Northernblot, RNase protection assay, or in situ hybridization), analysis offatty acid composition, measurement of eicosanoids, and statisticalanalysis will be carried out, as described above in the context ofcultured cells.

Example 8 Transgenic Animals

The studies described here are designed to create transgenic mice thatglobally express the fat-1 gene and to characterize the tissue and organlipid profiles of these animals. Transgenic mice have become a valuablemodel for evaluation of physiological significance of a gene in vivo.Availability of transgenic mice allows us to study the effect of atransgene in a variety of cell types at different stages of an animal'slifespan. This n-3 transgenic mouse model will provide new opportunitiesto elucidate the roles of n-3 PUFA and compounds derived from them inthe development and cell biology.

To generate transgenic animals that can globally express the fat-1 gene,one can use an expression vector that contains a fat-1 gene and thechicken beta-actin promotor with the CMV enhancer (CAG promotor), whichis highly active in a wide range of cell types and therefore allowshigh-level and broad expression of the transgene (Niwa et al., Gene108:193–199, 1991; Okabe et al., FEBS Lett. 407:313–319, 1997). Theexpression construct will be microinjected into the pronuclei ofone-cell embryos of C57BL/6×C3H mice to produce transgenic mice. Theywill be bred and transgenic mouse line is established. Weanling mice arefed either a normal diet or a diet high in n-6 PUFA. Various tissueswill be harvested from these animals at different ages (neonate, wean—1month, adult—6 ms and aging—12 ms, 3–5 mice per time point will be used)for assessment of the expression levels of the transgene anddetermination of fatty acid composition. The levels of eicosanoids inplasma and various tissues will also be measured. A group of wild-typemice (C57BL/6) fed with the same diet (either a normal diet or a highn-6 diet) will be used as controls. The results will be compared withthose from wild type animals fed the same diet. The procedure isillustrated in FIG. 8.

The transgene will be prepared by methods similar to those described byOkabe et al. (supra). Briefly, a cDNA encoding the fat-1 gene isamplified by PCR with primers, 5-agaattcggcacgagccaa gtttgaggt-3′ (SEQID NO:1) and 5′-gcctgaggctttatgcattcaacgcact-3′ (SEQ ID NO:2), usingpCE8-fat1 (provided by Dr. J. Browse, Washington State University) as atemplate. No additional amino acid sequence is added on either side ofthe fat-1. The PCR product will be confirmed by DNA sequencing. TheEcoR1 and Bgl-II sites included in the PCR primers are used to introducethe amplified fat-1 cDNA into a pCAGGS expression vector containing thechicken beta-actin promoter and cytomegalovirus enhancer, beta-actinintron and bovine globin poly-adenylation signal (provided by Dr. JMiyazaki, Osaka University Medical School). The entire insert with thepromoter and coding sequence will be excised with BamHI and Sal1 andgel-purified.

Transgenic mouse lines will be produced by injecting the purified BamHIand SalI fragment into C57BL/6×C3H fertilized eggs. The DNA-injectedeggs are transplanted to pseudo-pregnant mice (B6C3F1) to producetransgenic mice. The founder transgenic mice will be identified by PCRand Southern blot analyses of tail DNA and bred with C57BL/6J mice.Offspring (either heterozygote or homozygote) will be used dependent onthe expression levels of the transgene or phenotype.

Weanling transgenic mice will be fed either a normal diet or a diet highin n-6 PUFA (see above). Animals will be sacrificed at different ages(neonate, wean to 1 month, adult to 6 mos and aging—12 mos, 3–5 mice pertime point will be used) and various tissues will be harvested forassessment of the expression level of transgene and determination offatty acid composition. The results will be compared with those fromwild type animals fed the same diet.

Other methods, including assessment of transgene expression (Northemblot, RNase protection assays, or in situ hybridization), analysis offatty acid composition, measurement of eicosanoids, and statisticalanalyses will be carried out as described above.

Example 9 Inhibition of Neuronal Cell Death

Construction of Recombinant Adenovirus (Ad): A recombinant Ad caryingthe fat-1 gene was constructed as described previously (Kang et al.,Proc. Natl. Acad. Sci. USA 98;4050–4054, 2001; see also, above). The n-3fatty acid desaturase cDNA (fat-1 gene) used was that described above,provided in plasmid pCE8. The fat-1 cDNA was excised from the plasmidwith an EcoRI/KpnI digestion, and inserted into pAdTrack-CMV vector. Theconstruct was subsequently recombined homologously with an adenoviralbackbone vector (pAdEasy 1) to generate two clones: Ad-GFP, whichexpresses GFP as a reporter or marker, and Ad-GFP-fat-1, which carriesboth the fat-1 and the GFP genes, each under the control of separate CMVpromoters. Recombinant adenoviral vector DNA was digested with PacI. Thelinerized vector DNA was mixed with SuperFect™ (QIAGEN) and used toinfect 293 cells. The recombinant viruses were prepared as high-titerstocks through propagation in 293 cells. The integrity of the constructswas confirmed by enzymatic digestion (i.e., restriction mapping) and byDNA sequencing. Purified virus was checked and its sequence confirmedagain by PCR analysis.

Tissue Culture and Infection with Ad: Rat cortical neurons were preparedusing standard techniques. Briefly, prenatal embryonic day 17 (E17) ratcortical neurons were dissociated and plated in poly-lysine-coated wellsat 2×10⁶ cells/well. The cells were grown in Neurobasal™ Medium (NBM,Life Technologies) supplemented with 25 μM glutamic acid (Sigma ChemicalCo., St. Louis, Mo.), 0.5 mM glutamine, 1% antibiotic-antimycoticsolution, and 2% B27 (Life Technologies). Cultures were kept at 37° C.in air with 5% CO₂ and 98% relative humidity. The culture medium waschanged every four days. After 8–10 days in culture, cells weretransfected with either the Ad-GFP (control) or the Ad-GFP-fat-1plasmids. Viral infections were carried out by adding viral particles tothe culture medium. After a 48-hour incubation, cells were used foranalyses of gene expression, fatty acid composition, eicosanoidproduction, and induction of apoptosis.

RNA Analysis: The level of fat-1 expression was determined by probingfor mRNA transcripts in an RNAse protection assay using the RPA III™ kit(Ambion, Austin, Tex.). Briefly, total RNA was extracted from culturedcells using a total RNA isolation reagent (TRizol, GIBco BRL) accordingto the manufacturer's protocol. The plasmid containing the fat-1 gene,pCE8, was linearized and used as a transcription template. Antisense RNAprobes were transcribed in vitro using [³³P]-UTP, T7 polymerase(Riboprobe System™ T7 kit, Promega), hybridized with total RNA (15 μg)extracted from neurons, and digested with ribonuclease to removenonhybridized RNA and probe. The protected RNA-RNA hybrids were resolvedin a denaturing 5% sequence gel and subjected to autoradiography. Aprobe targeting the β-actin gene was used as an internal control. fat-1mRNA was not detected in cells infected with Ad-GFP (control), but washighly abundant in cells infected with Ad-GFP-fat-1.

The cells were also examined by fluoresence microscopy. Infected cellsthat expressed the fat-1 gene were readily identifiable because theyco-expressed GFP. Forty-eight hours after infection, 30–40% of theneurons were infected and expressed GFP. These results demonstrate thatAd-mediated gene transfer confers high expression of fat-1 gene in ratcortical neurons, which normally lack the gene.

Lipid Analysis: The fatty acid composition of total cellular lipids wasanalyzed as described in Kang et al. (Proc. Natl. Acad. Sci. USA98:4050–4054, 2001). Lipid was extracted with chloroform:methanol (2:1,vol:vol) containing 0.005% butylated hydroxytoluene (BHT, as anantioxidant). Fatty acid methyl esters were prepared using a 14%(wt/vol) BF3/methanol reagent. Fatty acid methyl esters were quantifiedwith GC/MS by using an HP-5890 Series II gas chromatograph equipped witha Supelcowax™ SP-10 capillary column (Supelco, Bellefonte, Pa.) attachedto an HP-5971 mass spectrometer. The injector and detector aremaintained at 260° C. and 280° C., respectively. The oven program ismaintained initially at 150° C. for 2 minutes, then ramped to 200° C. at10° C./minute and held for 4 minutes, ramped again at 5° C./minute to240° C., held for 3 minutes, and finally ramped to 270° C. at 10°C./minute and maintained for 5 minutes. Carrier gas-flow rate ismaintained at a constant 0.8 ml/min throughout. Total ion monitoring isperformed, encompassing mass ranges from 50–550 atomic mass units. Fattyacid mass is determined by comparing areas of various analyzed fattyacids to that of a fixed concentration of internal standard.

The expression of fat-1 resulted in conversion of n-6 fatty acids to n-3fatty acids, and thus a significant change in the ratio of n-6:n-3 fattyacids. The fatty acid profile obtained from control cells issignificantly different from that of cells infected with Ad-GFP-fat-1(FIG. 9; see also FIG. 10). Cells infected with Ad-GFP show no change infatty acid composition when compared with non-infected cells. In cellsexpressing the n-3 desaturase, almost all types of n-6 fatty acids wereconverted to the corresponding n-3 fatty acids, namely, 18:2n-6 to18:3n-3, 20:4n-6 to 20:5n-3, 22:4n-6 to 22:5n-3, an 22:5n-6 to 22:6n-3.The change in fatty acid composition of the cells expressing the fat-1gene resulted in reduction of the n-6:n-3 ratio from 6.4:1 in thecontrol cells to 1.7:1 in the cells expressing the n-3 desaturase.Expression of the C. elegans n-3 fatty acid desaturase resulted in asignificant increase in the levels of DHA in transfected cells. Anincrease in levels of EPA and ALA is observed with a concomitantdecrease in AA and LA suggesting that the decrease in production of PGE₂resulted from both the shift in the n-6:n-3 fatty acid ratio and fromDHA-mediated inhibition of AA hydrolysis.

Measurement of Eicosanoids: 2-series eicosanoids may be associated withneuronal apoptosis in age-associated neurodegenerative diseases andacute excitotoxic insults such as ischemia (Sanzgiri et al., J.Neurobiol. 41:221–229, 1999; Drachman and Rothstein, Ann. Neurol.48:792–795, 2000; Bezzi et al., Nature 391:281–285, 1998). Arachidonicacid (AA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3) are theprecursors of 2- and 3-series of eicosanoids, respectively. To determinewhether the gene transfer-mediated alteration in the contents of AA andEPA may lead to a difference in the production of eicosanoids in thecells, we measured the production of prostaglandin E₂, one of the majoreicosanoids derived from AA, in infected cells after stimulation withcalcium ionophore A23187. More specifically, prostaglandin E₂ wasmeasured by using enzyme immunoassay kits (Cayman Chemical, Ann Arbor,Mich.) following the manufacturer's protocol. (The crossreactivity withprostaglandin E3 is 16%.) Cultured cells were washed with LH buffer(with 1% BSA) and incubated with the same buffer containing the calciumionophore A23187 (5 μM). After a 10-minute incubation, the conditionedbuffer was recovered and subjected to eicosanoid measurement. The amountof prostaglandin E₂ produced by fat-1 expressing cells was 20% lowerthan that produced by control cells (FIG. 11).

Induction of apoptosis and determination of cell growth and viability:Apoptosis was induced by growth factor withdrawal. Forty-eight hoursafter neurons were transfected, the culture media was changed toNeurobasal™ Medium supplemented with 25 mM glutamic acid (Sigma ChemicalCo., St. Louis, Mo.) and 0.5 mM glutainine. Cytotoxicity was measured 24hours after growth factors were withdrawn using the Vybrant™ ApoptosisAssay (Molecular Probes, Eugene, Oreg.). Briefly, cells were washed withice-cold phosphate buffered saline (PBS) and subsequently incubated onice for 20–30 min in ice-cold PBS containing Hoechst 33342 solution (1ml/ml) and PI solution (1 ml/ml). A photograph was taken at the end ofthe incubation period.

Cell growth and viability: Cell growth and viability were determinedusing the MTT cell proliferation kit (Roche Diagnostic Corporation). MTTlabeling reagent (100 μl) was added to each well. After 4 hours ofincubation, 1.0 ml of the solubilization solution was added into eachwell. The cells were then incubated overnight at 37° C. and thespectrophotometrical absorbency of the solution at 600 nm was measured.

Expression of the fat-1 gene provided strong protection againstapoptosis in rat cortical neurons. Hoest 33625 and PI staining ofcortical cultures 24 hours after the induction of apoptosis, show thatcultures infected with Ad-GFP-fat-1 underwent less apoptosis than thoseinfected with Ad-GFP. MTT analysis indicated that the viability ofAd-GFP-fat-1 cells was significantly (p<0.05) higher than that of cellsinfected with Ad-GFP (FIG. 12). These results indicate that theexpression of fat-1 can inhibit neuronal apoptosis and promote cellviability. The ability of the C. elegans n-3 fatty acid desaturase toinhibit apoptosis of neuronal cells highlights the importance of then-6:n-3 fatty acid ratio in neuroprotection. Accordingly, techniquesthat deliver a fat-1 sequence, or a biologically active variant thereof,to neurons provide the means to quickly and dramatically balancecellular n-6:n-3 fatty acid ratio, alter eicosanoid profile (and therebyexert an anti-apoptotic effect on neuronal cells) without the need forsupplementation with exogenous n-3 PUFAs. Compared to dietaryintervention, this approach is more effective in balancing the n-6:n-3ratio because it simultaneously elevates the tissue concentration of n-3PUFAs and reduces the level of endogenous n-6 PUFAs. This method is anovel and effective approach to modifying fatty acid composition inneuronal cells, and it can be applied as a stand-alone gene therapy oras an adjuvant therapy or chemopreventive procedure (in, for example,apoplexy patients).

Data analysis, statistical analysis: Cell viability data (MTT), as wellas fatty acid composition and eicosanoids levels were compared using theStudent t-test. The analysis included 6 wells/group (except lipidanalyses; 4 wells/group) and each experiment was repeated 3 times. Thelevel of significance was set at p<0.05.

Example 10 Fat-1 Expression in Human Endothelium and Inhibition ofInflammation

To determine whether the conversion of n-6 to n-3 PUFA can begenetically conferred to primary human vascular endothelium and to studyits potential protective effects against endothelial activation aftercytokine stimulation, a first generation (type 5) recombinant adenoviralvector (Ad) was constructed which contained the fat-1 transgene inseries with a GFP expression cassette under the control of the CMVpromoter (Ad.fat-1). A GFP/β-gal adenovirus served as the control vector(Ad.GFP/β-gal). Monolayers of primary human umbilical vein endothelialcells (HUVECs) were infected with Ad.fat-1 or the control Ad for 36hours, exposed for 24 hours to 10 mM arachidonic acid, and subjected tolipid analysis by gas chromatography, surface adhesion molecule analysisby inimunoassay, and videomicroscopy to study endothelial interactionswith the monocytic cell line, THP-1, under laminar flow conditions.

Expression of fat-1 dramatically altered the lipid composition of humanendothelial cells and changed the overall ratio of n-6 to n-3 PUFA from8.5 to 1.4. Furthermore, after cytoline exposure (TNF-α, 5 μl appliedfor 4 hours) fat-1 expression significantly reduced the surfaceexpression of the adhesion molecules and markers of inflammation(E-Selectin, ICAM-1, and VCAM-1 by 42%, 43%, and 57%, respectively(p<0.001)).

We then asked whether changes in the adhesion molecule profile weresufficient to alter endothelial interactions with monocytes, the mostprevalent white blood cell type found in atherosclerotic lesions. Underlaminar flow and a defined shear stress of ˜2 dynes/cm², fat-1-infectedHUVEC, compared to control vector-infected HUVEC, supported ˜50% lessfirm adhesion with almost no effect on the rolling interactions of THP-1cells. Thus, heterologous expression of the C. elegans desaturase,fat-1, confers on human endothelial cells the ability to convert n-6 ton-3 PUFA. This effect significantly repressed cytokine induction of theendothelial inflammatory response and firm adhesion of the monocyticcell line, THP-1, under simulated physiological flow conditions.Accordingly, expression of fat-1 represents a potential therapeuticapproach to treating inflammatory vascular diseases, such asatherosclerosis.

Example 11 n-3 Desaturase as an Anti-Arrhythmic Agent

To determine whether fat-1 expression could provide an anti-arrhythmiceffect, myocytes expressing the n-3 desaturase were examined for theirsusceptibility to arrhythmias induced by arrhythmogenic agents. Neonatalrat cardiac myocytes, grown on glass coverslips and able tospontaneously beat, were infected with Ad.GFP.fat-1 or Ad.GFP. Two daysafter infection, cells were transferred to a perfusion system andperfused with serum free medium containing high concentrations (5–10 mM)of calcium. These media are arrhythmogenic. During the perfusionprocess, myocyte contraction was monitored using a phase contrastmicroscope and video-monitor edge-detector. Following the high [Ca²⁺](7.5 mM) challenge, the control cells infected with Ad.GFP promptlyexhibited an increased beating rate followed by spasmodic contractionsor fibrillation whereas the cells infected with Ad.GFP.fat-1 couldsustain regular beating. Thus, myocytes expressing the n-3 desaturaseshow little, if any, susceptibility to arrhythmogenic stimuli (FIG. 13).

Example 12 Fat-1 Expression and Inhibition of Tumor Growth

To test the effect of the gene transfer on tumor growth in vivo, we havecarried out a pilot experiment in two nude mice bearing human breastcancer xenografts (MDA-MB-231). One mouse was injected intratumorallywith 50 ml of Ad.GFP.fat-1 (1012 particles/ml) twice every other day.The other was injected with the control vector (Ad.GFP). The growth rateof the tumors was monitored for four weeks. The growth rate of the tumortreated with Ad.GFP.fat-1 appeared to be slower than that of the controltumor (FIG. 14).

Example 13 The Effect of Fat-1 Expression on Fatty Acid Composition andGrowth of Human Breast Cancer Cells in Culture

Construction of Recombinant Adenovirus (Ad): A recombinant Ad carryingthe fat-1 cDNA was constructed as described previously (Kang et al.,Proc. Natl. Acad. Sci. USA 98:4050–4054, 2001). Briefly, the fat-1 cDNAin pCE8 (as described above) was excised from the plasmid with anEcoRI/KpnI double digest, inserted into a shutter vector and thensubjected to homologous recombination with an adenoviral backboneaccording to the methods of He et al. (Proc. Natl. Acad. Sci. USA95:2509–2514, 1998). Two first-generation type 5 recombinantadenoviruses were generated: Ad.GFP, which carries GFP as a reportergene under control of the CMV promoter, and Ad.GFP.fat-1, which carriesboth the fat-1 and GFP genes, each under the control of separate CMVpromoters. The recombinant viruses were prepared as high titer stocksthrough propagation in 293 cells, as described previously (Kang et al.,Proc. Natl. Acad. Sci. USA 98:4050–4054, 2001). The integrity of theconstructs was confirmed by enzymatic digestion and by DNA sequenceanalysis.

Cell Cultures and Infection with Ad.: MCF-7 cells were routinelymaintained in 1:1 (v/v) mixture of DMEM and Ham's F12 medium (JRH,Bioscience) supplemented with 5% fetal bovine serum (FBS) plusantibiotic solution (penicillin, 50 U/ml; streptomycin, 50 μg/ml) at 37°C. in a tissue culture incubator with 5% CO₂ and 98% relative humidity.Cells were infected with Ad for experiments when they were grown toabout 70% confluence by adding virus particles to medium without serum(3–5×10⁸ particles/ml). Initially, optimal viral concentration wasdetermined by using Ad.GFP to achieve an optimal balance of high geneexpression and low viral titer to minimize cytotoxicity. After a 24-hourincubation, the infection medium was replaced with normal culture mediumsupplemented with 10 μM 18:2n6 and 20:4n6. Forty-eight hours afterinfection, cells were used for analyses of gene expression, fatty acidcomposition, eicosanoid production, and cell proliferation andapoptosis.

RNA Analysis: The fat-1 transcripts were examined by ribonucleaseprotection assay using a RPA III™ kit (Ambion, Austin, Tex.). Briefly,total RNA was extracted from cultured cells using a RNA isolation kit(Qiagen) according to the manufacturer's protocol. The plasmidcontaining fat-1, pCE8, was linearized and used as transcriptiontemplate. Antisense RNA probes were transcribed in vitro using ³³P-UTPand T7 polymerase (Riboprobe™ System T7 kit, Promega), hybridized withthe total RNA extracted from the cancer cells, and digested with RNaseto remove non-hybridized RNA and probe. The protected RNA:RNA wasresolved in denaturing sequence gel and subjected to autoradiography. Aprobe targeting the GAPDH gene was used as an internal control.

The cells that were infected and expressed the transgene could bereadily identified by fluorescence microscopy since they co-expressedthe GFP (which exhibites bright fluorescence). Three days afterinfection, it was observed that about 60–70 percent of the cells wereinfected and expressed the transgene. Analysis of mRNA using aribonuclease protection assay showed that fat-1 mRNA was highly abundantin cells infected with Ad.GFP.fat-1, but was not detected in cellsinfected with Ad.GFP (control). This result indicates that theAd-mediated gene transfer could confer a high expression of fat-1 genein MCF-7 cells, which normally lack the gene.

Lipid Analysis: To examine the efficacy of the gene transfer inmodifying the fatty acid composition of the human MCF-7 cells, totalcellular lipids were extracted and analyzed by gas chromatograph afterinfection with the Ads and incubation with n-6 fatty acids for 2–3 days.The fatty acid composition of total cellular lipids was analyzed asdescribed (Kang et al., supra). Lipid was extracted withchloroform/methanol (2:1, vol/vol) containing 0.005% butylatedhydroxytoluene (BHT, as antioxidant). Fatty acid methyl esters wereprepared by using a 14% (wt/vol) BF3/methanol reagent. Fatty acid methylesters were quantified with GC/MS by using an HP-5890 Series II gaschromatograph equipped with a Supelcowax SP-10 capillary column(Supelco, Bellefonte, Pa.) attached to an HP-5971 mass spectrometer. Theinjector and detector are maintained at 260° C. and 280° C.,respectively. The oven program is maintained initially at 150° C. for 2min, then ramped to 200° C. at 10° C./min and held for 4 min, rampedagain at 5° C./min to 240° C., held for 3 min, and finally ramped to270° C. at 10° C./min and maintained for 5 min. Carrier gas-flow rate ismaintained at a constant 0.8 ml/min throughout. Total ion monitoring isperformed, encompassing mass ranges from 50–550 atomic mass units. Fattyacid mass is determined by comparing areas of various analyzed fattyacids to that of a fixed concentration of internal standard.

The expression of fat-1 cDNA in MCF-7 cells resulted in conversions ofn-6 fatty acids to n-3 fatty acids, and a significant change in theratio of n-6/n-3 fatty acids. The fatty acid profiles are remarkablydifferent between the control cells infected just with the Ad.GFP andthe cells infected with the Ad.GFP.fat-1 (FIG. 15). Cells infected withAd.GFP had no change in their fatty acid profiles when compared withnoninfected cells. In the cells expressing the fat-1 cDNA (n-3 fattyacid desaturase), various n-6 fatty acids were converted largely to thecorresponding n3 fatty acids, for example, 18:2n6 to 18:3n3, 20:4n6 to20:5n3, and 22:4n6 to 22:5n3. As a result, the fatty acid composition ofthe cells expressing fat-1 gene was changed significantly when comparedwith that of the control cells infected with Ad.GFP (FIG. 15), with alarge reduction of the n-6/n-3 ratio from 12 in the control cells to 0.8in the cells expressing the n-3 fatty acid desaturase.

Measurement of Eicosanoids: It has been shown previously thatprostaglandin E2 (PGE2), one of the major ecosanoids derived from 20:4n6(arachidonic acid), is associated with cancer development (Rose andconnolly, Pharmacol. ther. 83:217–244, 1999; cave, Breast Cancer Res.Treat. 46:239–246, 1997). To determine whether the gene transfer-inducedalteration in the contents of arachidonic and eicosapentaenoic acids canchange the production of eicosanoids in the cells, we measured theproduction of PGE2 in the infected cells after stimulation with calciumionophore A23187 by using an enzyme immunoassay kit that specificallydetects prostaglandin E2 derived from AA with a 16% crossreactivity withprostaglandin E3 from EPA. More specifically, prostaglandin E₂ wasmeasured by using enzyme immunoassay kits (Assay Designs, Inc) followingthe manufacturer's protocol. (The cross-reactivity with PGE₃ is 16%).Cultured cells were washed with PBS containing 1% BSA and incubated withserum-free medium containing calcium ionophore A23187 (5 μM). After a10-minute incubation, the conditioned medium was recovered and subjectedto eicosanoid measurement. The amount of prostaglandin E₂ produced bythe fat-1 cells was significantly lower than that produced by thecontrol cells (FIG. 16).

Analysis of Cell Proliferation and Apoptosis: To determine the effect ofexpression of the fat-1 gene on MCF-7 cell growth, cell proliferationand apoptosis following gene transfer were assessed. Routinely, cellmorphology was examined by microscopy (dead cells appear to be detached,round and small) and total number of cell in each well was determined bycounting the viable cells using a hemocytometer. In addition, cellproliferation was assessed using a MTT Proliferation Kit I (RocheDiagnostics Corporation). Apoptotic cells were determined by nuclearstaining with Vybrant™ Apoptosis Kit #5 (Molecular Probes) following themanufacturer's protocol.

A large number of the cells expressing fat-I gene underwent apoptosis,as indicated by morphological changes (small size with round shape orfragmentation) and nuclear staining (bright blue). Statistic analysis ofapoptotic cell counts showed that 30–50% of cells infected withAd.GFP.fat-1 were apoptotic whereas only 10% dead cells found in thecontrol cells (infected with Ad.GFP). MTT analysis indicated thatproliferative activity of cells infected with Ad.GFP.fat-1 wassignificantly lower than that of cells infected with Ad.GFP.Accordingly, the total number of viable cells in the cells infected withAd.GFP.fat-1 was about 30% less than that in the control cells. Theseresults are consistent with the proposition that fat-1 expression canserve as an anti-cancer agent.

Data analyses, statistical analyses: Data were presented as mean ±SE.Student's T test was used to evaluate the difference between two values.The level of significance was set at p<0.05.Results

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

1. A nonhuman transgenic mammal whose genome comprises a nucleic acidmolecule comprising a nucleotide sequence operably linked to a promoter,wherein the nucleotide sequence encodes the amino acid sequence of SEQID NO:4 or an amino acid sequence that is (i) a biologically activevariant of SEQ ID NO:4 and (ii) at least 90% identical to SEQ ID NO:4,and upon expression of the nucleotide sequence in cells within thetransgenic mammal, the n-3 polyunsaturated fatty acid (PUFA) content iselevated compared to that of cells of a wild-type mammal, wherein thetransgenic mammal is selected from the group consisting of a mouse, acow, a pig, a sheep, a goat, and a rabbit.
 2. The transgenic mammal ofclaim 1, wherein the amino acid sequence is at least 95% identical toSEQ ID NO:4.
 3. The transgenic mammal of claim 1, wherein the amino acidsequence is at least 99% identical to SEQ ID NO:4.
 4. The transgenicmammal of claim 1, wherein the nucleotide sequence encodes SEQ ID NO:4.5. The transgenic mammal of claim 1, wherein the nucleotide sequencecomprises SEQ ID NO:3.
 6. The transgenic mammal of claim 1, wherein thenucleotide sequence consists of SEQ ID NO:3.
 7. The transgenic mammal ofclaim 1, wherein the mammal is a cow.
 8. The transgenic mammal of claim1, wherein the mammal is a pig.
 9. The transgenic mammal of claim 1,wherein the mammal is a sheep.
 10. The transgenic mammal of claim 1,wherein the mammal is a goat.
 11. The transgenic mammal of claim 1,wherein the mammal is a rabbit.
 12. The transgenic mammal of claim 1,wherein the biologically active variant is at least 90% as efficient asSEQ ID NO:4 in converting n-6 to n-3 PUFA.
 13. The transgenic mammal ofclaim 1, wherein the biologically active variant is at least 95% asefficient as SEQ ID NO:4 in converting n-6 to n-3 PUFA.
 14. Thetransgenic mammal of claim 1, wherein the biologically active variant isat least 99% as efficient as SEQ ID NO:4 in converting n-6 to n-3 PUFA.15. A nonhuman transgenic mammal whose genome comprises a nucleic acidmolecule comprising a nucleotide sequence operably linked to a promoter,wherein the nucleotide sequence encodes an amino acid sequence that is(i) at least 90% as efficient as SEQ ID NO:4 in converting n-6 to n-3PUFA and (ii) at least 90% identical to SEQ ID NO:4, and upon expressionof the nucleotide sequence in cells within the transgenic mammal the n-3PUFA content is elevated compared to that of cells of a wild-typemammal, wherein the transgenic mammal is selected from the groupconsisting of a mouse, a cow, a pig, a sheep, a goat, and a rabbit. 16.A transgenic mouse whose genome comprises a nucleic acid moleculecomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 4 operably linked to a promoter, wherein the mouse exhibits anelevated n-3 PUFA content as compared to a wild-type mouse.